Int J Pharm Pharm Sci, Vol 7, Issue 10, 386-389Original Article
DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR ESTIMATION OF DARUNAVIR ETHANOLATE IN BULK AND TABLETS
HEMANT K. JAIN1*, UMAKANT S. JADHAV1
1Department of Quality Assurance Techniques, STES’s, Sinhgad College of Pharmacy, Vadgaon (Bk.), Pune 411041, Maharashtra, India
Email: hemantkjain2001@yahoo.co.in
Received: 08 Jul 2015 Revised and Accepted: 02 Sep 2015
ABSTRACT
Objective:A new precise, accurate, sensitive and robust RP-HPLC method was developed for estimation of darunavir ethanolate in bulk and tablets.
Methods:The chromatographic separation was achieved on Enable C18 column (250 × 4.6 mm, 5 µm) at an ambient temperature. The mobile phase consists of acetonitrile and 0.01M potassium acetate buffer, pH 5.1 (75:25 v/v) was at the flow rate 1 ml/min and UV detection was done at 268 nm.
Results:The method was linear over the concentration range of 40-90 µg/ml (r2= 0.998) of the drug. The percentage content was found in darunavir ethanolate 99.19±0.58 in tablets. The low value of the drug %RSD (0.11) indicates that reproducibility of this method. Low value of LOD and LOQ suggests the sensitivity of the method.
Conclusion:It can be concluded from the results that the proposed RP-HPLC method was found to be rapid, simple, accurate, robust and precise for the analysis of darunavir ethanolate in bulk and tablet dosage form. The developed method can be applied in routine analysis of this drug in the pharmaceutical industry.
Keywords:Darunavir ethanolate, Validation and RP-HPLC.
© 2016 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
INTRODUCTION
Darunavir ethanolate is an oral anti-retroviral agent which selectively inhibits the cleavage of Human immunodeficiency virus (HIV-1) encoded Gag-polyproteins in infected cell, thereby preventing the formation of mature virus. Darunavir ethanolate has robust interaction with the protease enzyme from many strains of HIV. It blocks HIV protease enzyme which is needed for HIV to multiply. Darunavir Ethanolate is chemically [(1S, 2R)-3-[[(4-aminophenyl) sulfonyl] (2-methylpropyl)amino]-2-hydroxy-1-(phenylmethyl) propyl]-carbamic acid (3R,3aS,6aR)-hexahydrofuro[2,3-b] furan-3-yl ester monoethanolate [1] (fig. 1). Literature survey revealed that some methods had been developed for determination of darunavir ethanolate by HPLC [2-4], HPTLC [5] and Spectrophotometric method [6]. Reported HPLC methods require more time for analysis of one sample and less number of samples may be analyzed. Therefore, the present work involves the development of a rapid RP-HPLC method for estimation of darunavir ethanolate in bulk and tablet dosage form and may be suitable for an industry as more number of samples can be analyzed in the given time period.
Fig. 1: Chemical structure of darunavir ethanolate
MATERIALS AND METHODS
Chemicals and reagents
The reference standard of darunavir ethanolate was provided by Cipla Ltd, Aurangabad, India “Daruvir®-300 mg” tablets were procured from local pharmacies. The acetonitrile (HPLC grade) and potassium acetate of (AR grade) was provided by hind media Lab Pvt. Ltd, Mumbai, India.
Instruments
Shimadzu LC2010 HPLC system, Elga Lab water (PURE LAB UHQ-II) water purification system for HPLC study. Shimadzu model AY-120 balance, Enable C18 column, LC solution software was used.
Preparation of standard stock solutions
Darunavirethanolate (100 mg) was transferred to 100 ml of the volumetric flask, dissolved in acetonitrile and diluted up to the mark. The standard stock solution was further diluted to obtain six different concentrations between 40-90µg/ml. The 20 µl volume of each solution was injected into the HPLC system under the optimized chromatographic conditions.
Assay of tablet formulation
20 tablets of “Daruvir®-300” were weighed correctly and crushed to fine powder using a glass mortar and pestle. A portion equivalent to about 100 mg of darunavir ethanolate was correctly weighed and transferred to 100 ml volumetric flask. The powder was dissolved in acetonitrile, sonicated for 10 min and diluted up to the mark. The solution was further diluted with acetonitrile and filtered using what man filter paper no. 41. The filtrate was diluted to obtain 70µg/ml of drug and injected (in triplicate) to HPLC system.
RESULTS AND DISCUSSION
Selection of detection wavelength
Analytical wavelength was selected by taking the absorption spectrum of darunavir ethanolate which gave λmax of darunavir ethanolate. A Stock solution of the drug was prepared in acetonitrile and a UV spectrum of 100 µg/ml solution of darunavir ethanolate was scanned in the range 200-400 nm. UV spectrum (fig. 2) showed maximum absorbance at 268 nm. Hence, wavelength 268 nm was selected for the further studies.
Optimization of chromatographic conditions
The HPLC method was optimized with a view to develop an assay method for darunavir ethanolate. Initially, different columns, different combinations of mobile phases such as methanol: water and Acetonitrile: Potassium Acetate in different proportions, were tried at different flow rates. Finally, A mobile phase consisting acetonitrile and 0.01M potassium acetate buffer, pH 5.1 (75:25 v/v) at the flow rate 1 ml/min was selected. The injection volume was 20 µl. The analysis was performed at an ambient temperature with UV detection at 268 nm. The Chromatogram of darunavir ethanolate is shown in fig. 3 and the optimized chromatographical conditions are mentioned in table 1. The retention time of the drug is 3.85 min under optimized chromatographical conditions.
Fig. 2: UV-Spectrum of darunavir ethanolate in acetonitrile (100µg/ml)
Table 1: Optimized chromatographic conditions
Parameters |
Details |
Mobile phase |
Acetonitrile: potassium acetate (75:25 v/v) (pH 5.1) |
Column |
Enable C18, 250 mm x 4.6 mm, 5 µm |
Flow rate |
1.0 ml/minute |
Detection |
UV at 268 nm |
Column oven temperature |
Ambient |
Injection volume |
20 µl |
Run time |
10 min |
Retention time (min.) |
3.85+0.02 |
Diluent |
Acetonitrile: potassium acetate (75:25 v/v) (pH5.1) |
Assay of tablet formulation
The value of % drug found to be 100.3±0.58 in assay study which is satisfactory. The results of assay study are shown in table 3.
Method validation [7-9]
Validation of an analytical procedure is the process by which it is established by laboratory studies that the performance characteristics of the procedure meet the requirements for the intended analytical application. The developed chromatographic method was validated for system suitability, linearity and range, accuracy, precision, and robustness as per ICH guidelines [10].
Linearity and range
Linearity was evaluated on a set of six standard solutions containing 40-90 μg/ml, for darunavir ethanolate. The relationship between peak area (as a dependent variable) and concentration of the drug in the solution (as an independent variable) was established by the simple linear regression method. Calibration curve was constructed by plotting an average peak area vs concentration as shown in fig. 4. The regression equation was obtained as a calibration curve.
Fig. 3: Chromatogram of darunavir ethanolate
Fig. 4: Calibration curve of darunavir ethanolate
Table 3: Results of assay of darunavir ethanolate
S. No. |
Sample solution concentration (µg/ml) |
Sample solution area |
Mean sample solution area |
%Drug found |
1 |
70 |
3188057 |
3182058 |
100.3±0.58 |
2 |
70 |
3180406 |
||
3 |
70 |
3177712 |
The value of R2 is found to be 0.9982, which indicates that the calibration curve is linear in the concentration range of 40-90 μg/ml.
Limit of detection (LOD) and limit of quantification (LOQ)
Five sets of known concentrations (40-90 μg/ml) were prepared. Calibration curves were plotted for each set. LOD and LOQ were calculated using the following formulae.
Where, SD = Standard Deviation of response
S = Average of the slope of the calibration curve
The LOD and LOQ were calculated as 0.234 µg/ml and 0.734 µg/ml, respectively, which indicate the method is sensitive.
Precision
(i) Repeatability
In repeatability, six standard solutions were prepared each having a concentration of 70µg/ml of darunavir ethanolate. The response of each of these solutions was measured and percentage relative standard deviation (% RSD) was calculated. The results are mentioned in table 4.
(ii) Intermediate precision
For intraday precision, nine different solutions were prepared across the intended range (60, 70 and 80 µg/ml) with three replicates of each, and their area was measured on the same day. For interday precision, nine different solutions were prepared across the intended range (60, 70 and 80 µg/ml) with three replicates of each, and their corresponding area was measured on three subsequent days. The results were reported in terms of the relative standard deviation (RSD) table 5(a) & 5(b).
Table 4: Results for method precision (repeatability)
Precision |
Amount (µg/ml) |
Area |
Mean area±SD |
%RSD |
Repeatability |
70 |
3144329 |
3169144+3289.592 |
0.0445 |
70 |
3179660 |
|||
70 |
3173584 |
|||
70 |
3174938 |
|||
70 |
3181435 |
|||
70 |
3160917 |
Table 5 (a): Intraday precision studies
Precision |
Amount (µg/ml) |
Area |
Mean area±SD |
%RSD |
Intra-day (n=3) |
60 |
2795891 |
2788966±6176.946 |
0.2214 |
60 |
2786981 |
|||
60 |
2784025 |
|||
70 |
3188057 |
3182058±5366.788 |
0.1686 |
|
70 |
3180406 |
|||
70 |
3177712 |
|||
80 |
3594261 |
3585630±12049.74 |
0.336 |
|
80 |
3571863 |
|||
80 |
3590765 |
Table 5 (b): Inter-day precision studies
Precision |
Amount (µg/ml) |
Area |
Mean area±SD |
%RSD |
Inter-day (n=3) |
60 |
2795890 |
2788985±6152.44 |
0.2205 |
60 |
2786981 |
|||
60 |
2784085 |
|||
70 |
3144329 |
3165858±18890.26 |
0.5966 |
|
70 |
3179660 |
|||
70 |
3173584 |
|||
80 |
3590352 |
3585867±8754.01 |
0.2441 |
|
80 |
3575779 |
|||
80 |
3591469 |
The value of % RSD in repeatability, intraday precision and intraday precision indicate that method is precise.
Table 6: Results of recovery study for darunavir ethanolate
Spike level |
Amount added (µg/ml) |
Area |
Amount recovered (µg/ml) |
% Recovery |
Mean % recovery±S. D |
% RSD |
80% |
72 |
3373533 |
72.72 |
101.01 |
100.33+0.583124 |
0.58 |
3351109 |
72.24 |
100 |
||||
3345048 |
72.10 |
100 |
||||
100% |
80 |
3579287 |
77.20 |
96.5 |
96.60+0.115902 |
0.11 |
3587541 |
77.38 |
96.73 |
||||
3582467 |
77.27 |
96.59 |
||||
120% |
88 |
3973756 |
85.78 |
97.47 |
97.19+0.275015 |
0.27 |
3962042 |
85.53 |
97.19 |
||||
3951109 |
85.29 |
96.92 |
Table 7: Robustness data for darunavir ethanolate
Factor |
Level |
%RSD |
A: Change in flow rate |
||
0.9 |
-1 |
0.3149 |
1 |
0 |
0.3147 |
1.1 |
+1 |
0.6244 |
B: Change in temperature |
||
24 ° C |
-1 |
0.2501 |
25 ° C |
0 |
0.2502 |
26 ° C |
+1 |
0.2471 |
C: Change in wavelength |
||
267 nm |
-1 |
0.7322 |
268 nm |
0 |
0.7279 |
269 nm |
+1 |
0.7229 |
Robustness
Robustness of the above method was carried out by purposefully varying some chromatographic method parameters. These parameters include changes in the flow rate (0.9 ml and 1.1 ml), temperature (24 °C and 26 °C) and wavelength (267 nm and 269 nm). The results obtained by changing these conditions are obtained in terms of % RSD values. The values % RSDs are given in table 7. These values are within acceptance an criterion which indicates that the developed method is robust.
Accuracy
Recovery studies were performed at 80.0%, 100.0% and 120.0% levels using the standard addition method. Standard drug solution of darunavir ethanolate was spiked at 80.0%, 100.0% and 120.0%. For each level, three replicate were prepared and injected in the column under optimized HPLC conditions.
The calculation for accuracy of darunavir ethanolate was performed and the results are expressed in terms of % recovery±SD and % R. S. D values (table 6). The values of % recovery at each level are found within acceptance criteria that indicate the method is accurate.
CONCLUSION
The present work represents the report that deals with analysis of darunavir ethanolate in bulk and tablet dosage forms using RP-HPLC. It can be concluded from the results that the proposed method is simple, accurate, robust and precise.
This method was validated as per ICH guidelines. Thus, it can be used for routine quality control studies for assay of darunavir ethanolate.
ACKNOWLEDGEMENT
Authors are grateful to Cipla Ltd, Aurangabad, India for providing API darunavir ethanolate as a gift sample. Authors also like to thanks principle of Sinhgad college of Pharmacy, Pune, India for providing necessary facilities to complete this project.
CONLFICT OF INTRESTS
Declared None
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